Biocontrol of Major Grapevine Diseases: Leading Research

1 Major Biocontrol Studies and Measures against Fungal and Oomycete Pathogens of Grapevine

A. ZANZOTTO

* AND

M. MORRONI

Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di ricerca per la Viticoltura, ConeglianoItaly

*Corresponding author: alessandro.zanzotto@entecra.it

Introduction

Grapevine and major fungal and oomycetal diseases

The productivity of grapevine cultivation around the world is challenged by the harmful action of many plant pathogens, including fungi, oomycetes, prokaryotes (bacteria, phytoplasmas), arthropods (insects, mites), nematodes and viruses.

In this review, we report on and discuss information from the available literature about the biological control of some major grapevine (Vitis vinifera L.) diseases caused by fungi or oomycetes:

•  downy mildew, caused by the oomycete Plasmopara viticola (Berk. & M.A. Curtis) Berl. and De Toni;

•  powdery mildew, caused by the fungus Erysiphe (syn. Uncinula) necator (Schwein.) Burrill;

•  grey mould, caused by the fungus Botrytis cinerea Pers.;

•  diseases of grapevine woody tissue caused by the fungi Eutypa lata (Pers.) Tul. and C. Tul., Botryosphaeriaceae spp., Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. and Mugnai) Crous and W. Gams, Phaeoacremonium spp. and Fomitiporia mediterranea M. Fisch, including esca disease of mature grapevines, which is associated with the latter three species.

Since the end of the Second World War, disease management has mostly been based on chemical pesticides (fungicides, insecticides, etc.), but the continuous use of these chemicals has raised widespread concern about their possible risks towards consumers, winegrowers and, also, bystanders. The collateral, negative effects of chemical pesticides on the environment were debated as well and have been the subject of recent European Union (EU) legislation (Directive 2009/128/EC, Regulation (EC) No. 1107/2009). The induction of resistance to the action of chemical pesticides in the pathogens, through repeated use of the same active ingredient, is another factor that has stimulated the use of alternative, non-chemical, control agents.

To support passage from a chemical-based disease control strategy towards sustainable agricultural practices, several microorganisms were tested for their ability to affect pathogen development in plant tissues. In addition, the mechanisms of some plant–pathogen interactions have been investigated in order to clarify the capacity of various chemical compounds to induce resistance reactions in the host plants.

Induced resistance

Resistance to pathogen infection can be limited to a few cells, as in localized acquired resistance (LAR), or it can be extended to other cells that are not directly affected by the pathogen, as in systemic acquired resistance (SAR) (Kessmann et al., 1994; Sticher et al., 1997). SAR is mediated by salicylic acid (SA) (or its chemical analogues) acting against a wide range of organisms and it is characterized by the expression of pathogenesis-related (PR) proteins (Durrant and Dong, 2004). In induced systemic resistance (ISR), the enhancement of plant defences is mediated by jasmonic acid (JA) and can be expressed after applications of non-pathogenic rhizobacteria, which are called plant growth-promoting rhizobacteria (PGPR) (Van Loon et al., 1998; Pieterse et al., 2001; Bakker et al., 2007) or other biocontrol agents (BCAs).

ISR plant defences are primed but not really activated until the infection occurs, and then higher levels of defence gene products, reactive oxygen species (ROS) and phenolic compounds are produced (reviewed in Gessler et al., 2011). Among these, resveratrol has an important role, both in plant defence and as a nutraceutical element. This compound has both pharmacological and antioxidant properties (Bavaresco et al., 2012). Comprehensive descriptions of the complex mechanisms that form the basis of the various plant defence systems have recently been given in reviews by Fu and Dong (2013) and Gozzo and Faoro (2013) for SAR, by Pieterse et al. (2014) for ISR and the defence priming system, and by Conrath (2011) for molecular aspects of the defence priming system.

Many studies have highlighted the role in plant disease resistance mechanisms of several substances and microorganisms, and they were eventually considered for exploitation in crop protection. The possibilities for the practical application of induced resistance in disease management are discussed by Walters et al. (2013).

In the review presented in this chapter, the various substances that have been reported as showing effects against plant pathogens and that could be considered as putative alternatives to chemical fungicides, have been divided into two main groups: inorganic compounds; organic chemical inducers and natural extracts – the latter being further subdivided into plant, fungal and compost extracts. The last part of the chapter looks specifically at biocontrol agents – fungi, yeast-like fungi, bacteria and oomycetes.

Inorganic Compounds

Copper is a common element used against downy mildew in viticulture. Additionally to its direct effect on the pathogen, a Bordeaux mixture, which is based on copper sulfate – and to a lesser extent a copper hydroxide formulation – increased the level of plant defence indicators such as peroxidases (POXs), phenols, resveratrol and anthocyanins (Coulomb et al., 1999). Aziz et al. (2006) demonstrated that copper sulfate can induce the synthesis of the phytoalexins cis- and trans-resveratrol, cis- and trans-e-viniferine and cis- and trans-piceid in leaves of the Chardonnay grapevine cultivar, effects that were increased by joint treatment with chitosan oligomers; chitosan oligomers alone reduced the diameter of B. cinerea lesions and the amount of leaf surface infected by P. viticola, and these effects were increased by the joint administration of copper sulfate.

Together with copper, Sulfur is a substance permitted in organic viticulture where it is used to control powdery mildew. Aluminium chloride, used in combination with seaweed extract in the commercial product Synermix®, has been shown to enhance resveratrol production in detached grapevine leaves and increase the efficiency of anti-Botrytis treatments in a field trial (Jeandet et al., 2000).

Potassium silicate (Bowen et al., 1992) and calcium, acting on the cell wall structure, have a role in increasing plant protection. Reynolds et al. (1996) reported that potassium silicate (K2SiO3) treatments of grapevine cv. Bacchus, reduced the incidence of powdery mildew in 2 out of 3 years of trials. Calcium chelates the cell wall pectic components of the host, reducing their degradation by B. cinerea, but without significant effect on the infection rate (Chardonnet et al., 1997). Chardonnet et al. (1999) also observed that calcium can have a direct effect on the pathogen’s cell walls. Miceli et al. (1999) reported that calcium application effectively controlled grey mould in bunches of vines grown outdoors, as well as in the cold room.

Nigro et al. (2006) obtained positive results in the control of B. cinerea on table grapes, using calcium chloride, sodium bicarbonate and other carbonate salts. Sodium bicarbonate was also tested by Karabulut et al. (2003) and found to effectively control postharvest diseases of grapes. Zerbetto et al. (2002, 2004), reporting results obtained in some vineyards treated with sodium bicarbonate and a mixture of sodium bicarbonate with paraffinic oil, observed that the treatment with sodium bicarbonate was ineffective against P. viticola and B. cinerea, while it positively protected the vineyard against E. necator in low-risk situations. With high disease pressure, better results were obtained with a mixture of sodium bicarbonate and paraffinic oil. Limited phytotoxic effects were observed on the leaves (0.25 to 2%), but interveinal necroses occurred at higher concentrations (6 and 9%).

The effective use of potassium bicarbonate against E. necator and P. viticola has also been reported by Sawant and Sawant (2008) and Dagostin et al. (2011), respectively, with some phytotoxic effect shown in the latter case. Positive results from trials using potassium bicarbonate against B. cinerea on grapevine have been presented by Di Martino et al. (2014) from field trials and Youssef and Ruffo Roberto (2014a), also of table grapes; in the latter case also with the use of potassium sorbate. A possible induction of resistance was hypothesized after the postharvest incidence of grey mould was reduced by potassium sorbate in 2 out of 3 years of trials (Feliziani et al., 2013). Effective control of grey mould on table grapes was also observed by Youssef and Ruffo Roberto (2014b) with three other salts (sodium silicate, iron sulfate and ammonium bicarbonate), also after preharvest and postharvest treatments.

Organic Chemical Inducers and Natural Extracts

Details of the use of these agents in the control of grapevine diseases are summarized in Table 1.1.

Table 1.1 The use of organic chemical inducers and natural extracts in the control of grapevine diseases.

Beta-aminobutyric acid (BABA) is an isomer of aminobutyric acid and a non-protein amino acid that occurs only rarely in plants; it acts as a resistance inducer in BABA-induced resistance (BABA-IR) mechanism (Jakab et al., 2001). Cohen et al. (1999) reported good activity of BABA against downy mildew on grapevine leaf discs, with sporulation reduced by 85–94%, and with higher concentrations able to protect potted plants. Field evaluation has been conducted on grapevine leaves of cvs Chardonnay and Cabernet Sauvignon; these cultivars were effectively protected against downy mildew when BABA or a mixture of BABA and different fungicides were used (Reuveni et al., 2001). Hamiduzzaman et al. (2005) indicated that protection against downy mildew induced by BABA was induced by the deposition of callose and the potentiation of PR (pathogenesis-related) gene expression. Dubreuil-Maurizi et al. (2010) observed that in grapevine leaves treated with BABA, there was a stronger production of ROS after P. viticola infection.

Chitosans are natural non-toxic polymers of b-1,4-linked d-glucosamine and N-acetyl-d-glucosamine produced by treating (deacetylating) chitin from crustacean shells and fungal cell walls with sodium hydroxide. Their biological activity is related to the size of the oligomer, degree of deacetylation and number of amino groups (Kauss et al., 1989; Hadwiger et al., 1994). Repka (2001) observed an accumulation of PR proteins after the treatment of grapevine cell suspension cultures with chitosan. New insights into the structure–activity relationships of chitosan have been recently presented by Sahariah et al. (2014). The in vitro activity of chitosan against B. cinerea on excised Chardonnay cultivar leaves was reported by Trotel-Aziz et al. (2006), and by Aziz et al. (2006), who also reported the activity of chitosan against P. viticola. In the first study, treatments of the leaves induced the activity of lipoxygenase, phenylalanine ammonia-lyase (PAL) and chitinase. The best protection was achieved at the dose of 75–100 mg l–1. In the second study, the authors compared the effects of treating the leaves with chitosan oligomers either alone or in combination with copper sulfate. Chitosan oligomers induced the stimulation of chitinase and glucanase activities, in addition to an accumulation of stilbene phytoalexins. When chitosan oligomers were used in combination with copper sulfate, higher production of phytoalexins and increased resistance were observed. The results also seemed to indicate that the action of chitosan is related to the induction of resistance rather than a direct effect on the pathogens.

Results obtained by Reglinski et al. (2010) supported the work of Trotel-Aziz et al. (2006) in indicating that chitosan acted in a concentration-dependent manner, but also reporting a direct antifungal activity. The same authors tested chitosan in combination with Ulocladium oudemansii for the control of grey mould in the vineyard, and demonstrated effective control under conditions of low disease pressure. Interestingly, the application of chitosan was correlated with an increased activity of POX, an enzyme catalysing reactions that strengthen plant cell walls. The application of a chitosan solution to vines showed both preventive and curative control of infection by B. cinerea (Amborabé et al., 2004). Iriti et al. (2009) observed a partial stomatal closure in bean leaves induced by chitosan, thus making it an antitranspirant compound. Alternative methods to control postharvest grey mould on table grapes have been reviewed by Romanazzi et al. (2012), and further research by Feliziani et al. (2013) described the effects of preharvest chitosan treatments using different commercial formulations of chitosan on the quality and postharvest decay of table grapes.

In field trials, chitosan effectively reduced the severity of infection by P. viticola (Romanazzi et al., 2014), but showed phytotoxicity at high doses (Dagostin et al., 2011). Iriti et al. (2011) reported an increase of total polyphenol content and antioxidant power in the berry tissues of a V. vinifera cultivar treated with Kendal-COPS® (a formulation based on chitosan, Cu and Mn), together with a reduction in powdery mildew severity on leaves and bunches. Van Aubel et al. (2014), using a complex molecule formed by the combination of oligopectin and oligochitosan, reported positive results in protection against powdery mildew.

A product named Chitogel® enhanced the development of cv. Chardonnay plantlets and inhibited the growth of B. cinerea (Ait Barka et al., 2004). Use of the products Elexa®, Chito Plant® and Armour-Zen® have been cited in reviews by Elmer and Reglinski (2006) and Romanazzi et al. (2012).

Jasmonic acid is a plant hormone, and this substance and its derivative, methyl jasmonate, are involved in the regulation of several plant processes (reviewed in Avanci et al., 2010). They are concerned with the activation of plant defence responses, such as the elicitation of several defence-related genes, and with the accumulation of PR proteins, callose deposition, hypersensitive response-like cell death and stilbene phytoalexin biosynthesis (Repka et al., 2004, 2013; Vezzulli et al., 2007; Faurie et al., 2009). Belhadj et al. (2006), after treating cv. Cabernet Sauvignon with methyl jasmonate, obtained an enhanced tolerance against powdery mildew.

Laminarin is a glucan made up of glucose units linked by glycosidic bonds, and is extracted from the brown seaweed Laminaria digitata (Vera et al., 2011). It has been reported to induce the activation of defence-related genes in grape cells, and to elicit resistance to B. cinerea and P. viticola (Aziz et al., 2003). Sulfated laminarin induces priming mechanisms for resistance (Trouvelot et al., 2008). Allègre et al. (2009) studied the effects of laminarin in the P. viticola–V. vinifera pathosystem and observed that the elicitor induced the closure of stomata but that this effect alone was not sufficient to protect the leaves from downy mildew. Romanazzi et al. (2014) tested the effectiveness of laminarin against downy mildew in field trials, and observed a lack of protection when it was used alone but better results when it was used in combination with copper salts at lower doses and Saccharomyces spp. extracts.

Oligogalacturonides (OGAs) are released from plant cell walls and are homopolymers of d-galacturonic acid (Reymond et al., 1995). They have the ability to induce resistance to B. cinerea in detached grapevine leaves in a dose-dependent relationship (Aziz et al., 2004), triggering many defence-related genes as well as increasing chitinase and glucanase activities. The mode of action of OGAs also involves oxidative burst and protein phosphorylation mechanisms. De Miccolis Angelini et al. (2009), in a short review, cited promising results obtained with plant-derived elicitors, such as oligogalacturonides and cellodextrins.

Rhamnolipids are glycolipid biosurfactants produced by bacteria (reviewed in Vatsa et al., 2010). Their protective effects against B. cinerea have been evaluated on cell suspension cultures and in vitro plantlets by Varnier et al. (2009). Ca²+ influx, mitogen-activated protein kinase (MAPK) activation, ROS production and the induction of defence gene expression and the hypersensitive response (HR) are involved in the resistance mechanism. They act as MAMPs (microbe-associated molecular patterns), in that they are recognized by plants and induce a defence response involving different signalling sectors according to the type of pathogen (Sanchez et al., 2012). A direct effect on the fungal pathogen concerned has also been identified on spore germination and mycelium growth (Varnier et al., 2009). Dagostin et al. (2011) observed good control of the severity of P. viticola infection in field trials with the commercial product ZONIX™.

Salicylic acid (SA) is a plant hormone implicated in the coordination of plant disease resistance, and induces PR proteins in grapevine (reviewed in Elmer and Reglinski, 2006). Tamm et al. (2011) reported that, under controlled conditions, SA reduced downy mildew incidence on leaves. A small reduction in downy mildew severity (max. 50%) was observed in field trials using 0.2% SA (Kast, 2000) but, as reported by Elmer and Reglinski (2006), a concentration higher than 2 mM may cause phytotoxicity, thus raising problems for its practical use.

Plant extracts

Reynoutria sachalinensis (giant knotweed) extracts induce defence reactions in various crops (Schmitt, 2006). Elmer and Reglinski (2006) reported positive results with a formulation of R. sachalinensis extract (Milsana®), against grapevine powdery mildew and grey mould, obtained by some researchers in Germany. Milsana® was also evaluated by Konstantinidou-Doltsinis et al. (2007) in vineyard trials in which a significant reduction of powdery mildew disease severity was observed; the product was more effective when applied at the early stages of disease development. The effectiveness of a new formulation of R. sachalinensis extract against E. necator in vineyard trials was reported by Ortugno et al. (2014). R. sachalinensis extract has recently been added under code P5 (host plant defence inducers – plant extracts) to the Fungicide Resistance Action Committee (FRAC) list (FRAC, 2015).

Lizzi et al. (1998) reported a positive effect of extracts of the brown seaweed Ascophyllum nodosum against P. viticola in laboratory and greenhouse experiments, in which it acted as a resistance inducer on grapevine leaves. Di Marco (2010) gave a preliminary report on the activity of the commercial product Marvita® (which is based on A. nodosum extract) against esca disease. As mentioned above, a formulation of seaweed extract and aluminium chloride induced resveratrol accumulation in detached grapevine leaves. In vineyard trials, the efficacy of iprodione against B. cinerea was strengthened by the use of Synermix® (Jeandet et al., 2000).

The antifungal activity of Melaleuca alternifolia (tea tree) components was investigated by Hammer et al. (2003). The effectiveness of some tea tree extracts against downy mildew under controlled conditions was reduced in vineyard applications (Dagostin et al., 2011). La Torre et al. (2014) observed a positive effect of a product containing 23.8% tea tree oil against downy mildew, but with lower effectiveness than that of a cupric reference product in a field trial test. M. alternifolia extract is also reported in the FRAC (2015) list under the code F7 (lipid synthesis and membrane integrity – proposed cell membrane disruption).

The essential oil from Viola odorata flowers has been tested in vitro against B. cinerea (Hammami et al., 2011a), who obtained a strong inhibition of the pathogen, even at low concentrations. Harm et al. (2011) tested the efficacy of Solidago canadensis extract as an inducer of a defence reaction against P. viticola in potted vines grown outdoors and obtained more than 80% protection.

Arnault et al. (2013) reported the preliminary results of tests with hydroalcoholic solutions (Salix alba, Equisetum arvense, Artemisia vulgaris) or aqueous solutions (Frangula alnus, Rheum palmatum) of extracts of various plant species against P. viticola, in field conditions. Three products (from F. alnus and E. arvense, fructose solutions), combined with copper at 100g ha–1 dose, were as effective as copper alone at 600 g ha–1; no effects were reported with the A. vulgaris solution. Godard et al. (2009) evaluated the effectiveness of Rheum palmatum roots and Frangula alnus bark extracts, reporting fungitoxic and defence reaction effects against P. viticola on leaves and increased stilbenic phytoalexins and POX activity.

The antifungal activity of a Salvia officinalis (sage) alcoholic extract against P. viticola, on potted grapevine plants, was investigated by Dagostin et al. (2010). The extract gave effective control on the potted plants but in field trials its efficacy was lower, as its persistence was reduced by rainfall. In a large-scale study, Dagostin et al. (2011) also investigated the use of Yucca schidigera extract against downy mildew, and considered it to be worth further development. Another extract tested was that of Inula viscosa; this provided good control over P. viticola in one out of two field trials, but caused widespread phytotoxicity. Cohen et al. (2006), after growth chamber tests, reported that an oily paste extract of I. viscosa applied to grapevine leaves was effective in controlling downy mildew infections.

Fungal extracts

An extract of Penicillium chrysogenum was reported to control downy and powdery mildew as copper and sulfur fungicides or resistance inducers, under greenhouse and vineyard conditions (Thuerig et al., 2006). In certain conditions phytotoxic side-effects were observed. Since the fungal extract had no direct fungicidal effects, its protecting properties are likely to depend on the activation of defence mechanisms. Also Harm et al. (2011) tested the effectiveness of P. crysogenum extracts against downy mildew on grapevine, observing a minimal protection but the induction of a wide range of resistance-related metabolites.

A commercial formulation containing Saccharomyces cerevisiae extracts is available with the name of K&A Oomisine® to stimulate the natural control of P. viticola on grapevine. A new product containing cerevisane, an inert fraction from a selected strain of S. cerevisiae, gave interesting results against downy mildew, powdery mildew and grey mould after vineyard trials carried out in Italy (Pujos et al., 2014).

Compost extracts

The effect of water extracts of fermented composts from animal and plant sources (cattle manure, chicken–cattle manure and grape marc compost) was tested by Elad and Shtienberg (1994), who observed the reduction of B. cinerea infections on grape berries in growth chamber experiments. Sackenheim et al. (1994) observed a positive result in the control of P. viticola on grapevine cuttings in a growth chamber after the application of aqueous extracts of composted microbiologically active substrates.

The use of grape marc extract to elicit plant defence responses has been recently investigated by Goupil et al. (2012). Treatments of tobacco leaves with marc extracts of red V. vinifera cultivars induced HR-like effects with local and systemic up-regulation of PR1 and PR2 encoding genes. On the other hand Thuerig et al. (2011) studied the possibility of a site-specific resistance modulation by the application of organic amendments, but they did not observe any difference in leaf susceptibility to P. viticola.

Biocontrol Agents (BCAs)

Plants interact with many microbial organisms living inside the plants, as endophytes, or on the external surface, as epiphytes. Special attention has been given to fungi and bacteria, acting as biocontrol agents, plant growth-promoting agents and resistance inducers.

Fungi

Details of the use of fungi in the biocontrol of grapevine diseases are summarized in Table 1.2.

Table 1.2 Fungal biocontrol agents (BCAs) for the treatment of grapevine diseases.

Acremonium spp.: A. byssoides is an endophytic fungus, isolated from grapevine leaves by Burruano et al. (2008), who evaluated its activity against P. viticola. The biocontrol agent (BCA) parasitized the pathogen, with invasion of the mycelium and deformation of the asexual structures. Culture filtrates and extracts from A. byssoides completely inhibited the development of P. viticola sporangia. A. cephalosporium has been tested as a BCA against Botrytis, Aspergillus and Rhizopus rots by Zahavi et al. (2000).

Alternaria spp.: Fungi belonging to this genus are ubiquitous. A. alternata has been observed to inhibit the sporulation of P. viticola on grapevine leaves (Musetti et al., 2006) and the production of secondary metabolites, such as diketopiperazines, has also been reported (Musetti et al., 2007).

Ampelomyces quisqualis: This species includes many strains that are natural antagonists of powdery mildews, such as E. necator (Kiss, 1998), although it should be noted that it has been concluded after a series of molecular studies that A. quisqualis should not be considered as a single species, but as a species complex (Kiss and Nakasone, 1998). More recently, Angeli et al. (2012) also observed the existence of different physiological forms within the A. quisqualis species. The parasitization of E. necator cleistothecia and mycelium by A. quisqualis has been reported by Falk et al. (1995). One strain – M10 – has been commercialized as AQ10® and has been tested in vineyards, with good results under low–medium disease pressure and in alternation with systemic fungicides. When used in alternation with sulfur under high disease pressure, the efficacy was significantly lower (Monchiero et al., 1996; Zanzotto et al., 2005). In some experiments, late treatments with A. quisqualis caused a reduction in the production of E. necator chleistothecia (Zanzotto et al., 2005; Caffi et al., 2010; Legler et al., 2011).

Aphanocladium album: A. album is a BCA that both expresses a mycoparasitic activity and produces chitinolytic enzymes (Kuntz et al., 1992). Ciccarese et al. (2006) observed good activity when it was applied to grapes preharvest against rot diseases caused by B. cinerea and other microbial agents.

Chaetomium spp.: The in vitro activity of this ascomycete was preliminarily reported by Spagnolo et al. (2012) in the biocontrol of grapevine trunk diseases. The strain Cha1, isolated from asymptomatic trunk wood, was able to overgrow colonies of two Neofusicoccum parvum and Diplodia seriata strains and one strain each of both Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

Epicoccum nigrum: The effectiveness of this BCA against downy mildew was investigated by Kortekamp (1997), who eventually questioned the success of biological control by this species. E. nigrum gave good performances against B. cinerea (reviewed in Elmer and Reglinski, 2006). Suppression of infection and sporulation on detached rachii was observed, but field tests on the capacity of this BCA to reduce B. cinerea overwintering inoculum gave weak and inconsistent results (Fowler et al., 1999). The saprophytic activity of E. nigrum is considered to be its primary mode of action, but an effect due to the production of antimicrobial metabolites has also been hypothesized (reviewed in Elmer and Reglinski, 2006).

Fusarium proliferatum: F. proliferatum has been reported as an integrative BCA against grapevine downy mildew (Falk et al., 1996); multi-year vineyard treatments on two susceptible Vitis interspecific hybrids significantly reduced infection severity in some years. F. proliferatum was more effective in reducing disease severity than disease incidence. The authors considered that it might be used together with other products, such as sulfur or copper, on poorly susceptible varieties or in areas where the disease is not severe. A cold-tolerant strain of F. proliferatum (isolate 1505) produced through UV mutagenesis (Bakshi et al., 2001) was demonstrated to be capable of controlling P. viticola on detached grapevine leaves. Pruning wounds treated with Fusarium lateritium 1–14 days before artificial infection by Eutypa lata reduced recovery of the pathogen (John et al., 2005).

Gliocladium roseum (now reclassified as Clonostachys rosea): Sutton et al. (1997) reviewed the activity of this BCA against B. cinerea on various crops. Elmer and Reglinski (2006) reviewed two studies that evaluated the activity of members of Gliocladium spp. against B. cinerea on grapes. Both were published in 1998, but reported contrasting results. The mode of action of Gliocladium spp. as a BCA has been studied on different crops, and the results indicated antibiosis and mycoparasitism as possible mechanisms of action.

Trichoderma spp.: Trichoderma spp. are soil inhabiting ascomycota that are distributed worldwide, and colonize various substrates as well as the plant rhizosphere, acting as antagonists of several plant pathogens (reviewed in Verma et al., 2007; Sawant, 2014). Their biocontrol mechanism is based on the production of lytic enzymes and other secondary metabolites, and has been reviewed in Reino et al. (2008). Trichoderma spp. have positive effects on their host, enhancing growth and inducing plant defence responses (Harman, 2006; Vinale et al., 2008); Woo et al. (2006) reported that enzymes produced by the species cause the release of low molecular weight oligosaccharides that induce resistance.

In recent decades, a wide variety of Trichoderma spp. has been proposed as biocontrol agents in agriculture, and some strains have given promising results against B. cinerea, which have resulted in the formulation of commercial products, e.g. Trichodex®, based on T. harzianum (T39) (O’Neil et al., 1996; Elad, 2000). Strain T39 competes with the pathogen for nutrients, interfering with B. cinerea pectolytic enzymes and inducing resistance in the plant (reviewed in Elmer and Reglinski, 2006). T. harzianum (strains 1295-22 and P1) has proved to be effective against B. cinerea on grapes in multi-year field trials (Harman et al., 1996).

Trichoderma spp. have also been evaluated as BCAs against the grapevine trunk pathogens E. lata, Phaeomoniella chlamydospora, Phomopsis viticola and species of the Botryosphaeriaceae (Kotze et al., 2011). In particular, T. atroviride strain USPP-T1 was very effective in reducing infection by these pathogens in artificially inoculated pruning wounds. The utilization of T. harzianum as a BCA against E. lata had previously been evaluated by John et al. (2005) and Halleen et al. (2005), showing a good performance in the protection of pruning wounds. Pertot et al. (2009) gave a preliminary report of good control by T. atroviride (strain SC1) of esca disease agents on potted grapevines. Mutawila et al. (2011) applied Trichoderma spp. to grapevine pruning wounds as BCAs against trunk pathogens, and observed significant treatment × cultivar interactions in the incidence of Trichoderma in table and wine grapevines. The best time for application was 6 h after pruning, for both early and late vine pruning (Mutawila et al., 2013). Pajot et al. (2012) reported promising results obtained from testing T. atroviride strain I-1237 (EsquiveWP®) against canker diseases of grapevine. Reggiori et al. (2014) have recently described good results with the utilization of Remedier® (T. asperellum and T. gamsii) against esca disease in 4 years of experimentation in six Italian regions. Rootshield®, a commercial formulation based on the T. harzianum strain T22, gave good results in nurseries only if applied at the rooting stage (Di Marco and Osti, 2007).

The activity of T. harzianum T39 against downy mildew has been studied by Perazzolli et al. (2008), whose studies were subsequently integrated by the work of Palmieri et al. (2012), who investigated the relationships among T. harzianum T39, V. vinifera and P. viticola. They provided evidence of systemic resistance induced by the BCA and showed how a large number of proteins related to energy metabolism, redox signalling and stress are overexpressed in plants treated with T39 and infected by P. viticola. The effect of T39 on the expression of defence-related genes involved in contrasting P. viticola infections on different cultivars has been reported by Banani et al. (2014), suggesting an important role of the host genetic background in the level of biocontrol. After indoor and field